TIS7 interacts with the mammalian SIN3 histone deacetylase complex in epithelial cells

Article2 September 2002free access TIS7 interacts with the mammalian SIN3 histone deacetylase complex in epithelial cells Ilja Vietor Ilja Vietor Present address: Department of Histology and Molecular Cell Biology, Institute of Anatomy, Histology and Embryology, University of Innsbruck, Medical School, Muellerstrasse 59, A-6020 Innsbruck, Austria Search for more papers by this author Santhosh K. Vadivelu Santhosh K. Vadivelu IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Nikolaus Wick Nikolaus Wick IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Robert Hoffman Robert Hoffman IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Matt Cotten Matt Cotten IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Christian Seiser Christian Seiser Institute of Molecular Biology, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Irene Fialka Irene Fialka IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Winfried Wunderlich Winfried Wunderlich IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Astrid Haase Astrid Haase IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Gabriela Korinkova Gabriela Korinkova Department of Pediatrics, Faculty of Medicine, Palackeho University in Olomouc, Czech Republic Search for more papers by this author Gerald Brosch Gerald Brosch Institute of Microbiology, University of Innsbruck, Medical School, Innsbruck, Austria Search for more papers by this author Lukas A. Huber Corresponding Author Lukas A. Huber Present address: Department of Histology and Molecular Cell Biology, Institute of Anatomy, Histology and Embryology, University of Innsbruck, Medical School, Muellerstrasse 59, A-6020 Innsbruck, Austria Search for more papers by this author Ilja Vietor Ilja Vietor Present address: Department of Histology and Molecular Cell Biology, Institute of Anatomy, Histology and Embryology, University of Innsbruck, Medical School, Muellerstrasse 59, A-6020 Innsbruck, Austria Search for more papers by this author Santhosh K. Vadivelu Santhosh K. Vadivelu IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Nikolaus Wick Nikolaus Wick IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Robert Hoffman Robert Hoffman IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Matt Cotten Matt Cotten IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Christian Seiser Christian Seiser Institute of Molecular Biology, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Irene Fialka Irene Fialka IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Winfried Wunderlich Winfried Wunderlich IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Astrid Haase Astrid Haase IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria Search for more papers by this author Gabriela Korinkova Gabriela Korinkova Department of Pediatrics, Faculty of Medicine, Palackeho University in Olomouc, Czech Republic Search for more papers by this author Gerald Brosch Gerald Brosch Institute of Microbiology, University of Innsbruck, Medical School, Innsbruck, Austria Search for more papers by this author Lukas A. Huber Corresponding Author Lukas A. Huber Present address: Department of Histology and Molecular Cell Biology, Institute of Anatomy, Histology and Embryology, University of Innsbruck, Medical School, Muellerstrasse 59, A-6020 Innsbruck, Austria Search for more papers by this author Author Information Ilja Vietor2, Santhosh K. Vadivelu1, Nikolaus Wick1, Robert Hoffman1, Matt Cotten1, Christian Seiser3, Irene Fialka1, Winfried Wunderlich1, Astrid Haase1, Gabriela Korinkova4, Gerald Brosch5 and Lukas A. Huber 2 1IMP, Research Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria 2Present address: Department of Histology and Molecular Cell Biology, Institute of Anatomy, Histology and Embryology, University of Innsbruck, Medical School, Muellerstrasse 59, A-6020 Innsbruck, Austria 3Institute of Molecular Biology, Vienna Biocenter, University of Vienna, Vienna, Austria 4Department of Pediatrics, Faculty of Medicine, Palackeho University in Olomouc, Czech Republic 5Institute of Microbiology, University of Innsbruck, Medical School, Innsbruck, Austria ‡I.Vietor, S.K.Vadivelu and N.Wick contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4621-4631https://doi.org/10.1093/emboj/cdf461 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The mammalian SIN3 complex consists of histone deacetylases (HDAC1, HDAC2), several known proteins (SAP30, N-CoR) and as yet unidentified proteins. Here we show that the mouse tetradecanoyl phorbol acetate induced sequence 7 (TIS7) protein is a novel transcriptional co-repressor that can associate with the SIN3 complex. We have identified tis7 as a gene that is up-regulated upon loss of polarity in a mouse mammary gland epithelial cell line expressing an estrogen-inducible c-JunER fusion protein. In unpolarized cells, TIS7 protein levels increase and TIS7 translocates into the nucleus. Overexpression of tis7 causes loss of polarity and represses a set of genes, as revealed by cDNA microarray analysis. We have shown that TIS7 protein interacts with several proteins of the SIN3 complex (mSin3B, HDAC1, N-CoR and SAP30) by yeast two-hybrid screening and co-immunoprecipitations. TIS7 co-immunoprecipitated HDAC complex is enzymatically active and represses a GAL4-dependent reporter transcription. The transcriptional repression of endogenous genes by tis7 overexpression is HDAC dependent. Thus, we propose TIS7 as a transcriptional co-repressor affecting the expression of specific genes in a HDAC activity-dependent manner during cell fate decisions, e.g. scattering. Introduction The complex regulation of gene expression at the transcriptional level is controlled by the combinatorial action of sequence-specific DNA-binding proteins, transcriptional co-regulators and components of the basal transcriptional machinery (Chi et al., 1995; Goodrich et al., 1996; Mannervik et al., 1999). Acetylation of internal lysine residues of core histone N-terminal domains by histone acetyltransferases (HATs) and deacetylation by histone deacetylases (HDACs) are associated with transcriptional regulation (Hebbes et al., 1988; Braunstein et al., 1993). Several HATs and HDACs have been shown to also acetylate and deacetylate non-histone protein substrates (e.g. transcription factors), implicating internal lysine acetylation as a possible mechanism of rapid and reversible regulation (Kouzarides, 2000; Humphrey et al., 2001). The two major class I-containing histone deacetylation complexes are NuRD (nucleosomal remodelling and histone deacetylation) and SIN3 (reviewed in Knoepfler and Eisenman, 1999; Ahringer, 2000). The SIN3 complex shares four core proteins with NuRD (HDAC1, HDAC2, RbAp46 and RbAp48) and additionally contains Sin3, SAP18 and SAP30 (Ayer, 1999). RbAp46 and RbAp48 proteins interact with histones and histone deacetylases HDAC1 and HDAC2. The SIN3 complex does not bind to DNA directly, but through sequence-specific DNA-binding proteins: passive repressors, e.g. nuclear hormone receptors (Nagy et al., 1997). Sin3 proteins, mSin3A and mSin3B, act as adaptor molecules linking repressive transcription factors to accessory proteins. This facilitates targeting of the HDAC complexes (Hassig et al., 1997; Laherty et al., 1997; Knoepfler and Eisenman, 1999; Kyrylenko et al., 2000). However, SIN3-mediated repression is not exclusively HDAC dependent, suggesting that the SIN3 complex either contains an intrinsic repressor activity or it associates with other co-repressors (Laherty et al., 1997; Wong and Privalsky, 1998). To study transcriptional regulation during cell fate decisions, we have used the previously established c-JunER mammary epithelial system (Fialka et al., 1996). Activation of the estrogen-responsive c-JunER fusion protein results in reduced transepithelial resistance, disruption of intercellular junctions, their barrier function and in the formation of irregular multilayers. The reversible loss of epithelial polarity resembles a scattering phenotype with redistributed apical and basolateral proteins to the entire plasma membrane. Activation of c-JunER mimics the c-Jun activation in vivo and transcriptionally regulates a variety of AP-1 target genes (Fialka et al., 1996). In this study we identified, by RT–PCR-based differential display (DD RT–PCR), the TPA induced sequence 7 (tis7) gene (Varnum et al., 1989a) to be up-regulated upon the loss of epithelial polarity in c-JunER cells. TIS7, also known as PC4 (Tirone and Shooter, 1989) or IFRD1 (interferon related developmental regulator 1) (Buanne et al., 1998), was described previously as an immediate early gene (Tirone and Shooter, 1989; Varnum et al., 1989b; Guardavaccaro et al., 1994, 1995) that is possibly involved in the differentiation of various cell types (Tirone and Shooter, 1989; Guardavaccaro et al., 1994, 1995). However, the molecular function of TIS7 has not yet been elucidated. Here we present evidence that TIS7, up-regulated after c-Jun activation, translocates into the nucleus and acts as a transcriptional co-repressor. We identified by cDNA microarray analysis several genes specifically repressed by overexpressed TIS7. TIS7 can associate with the mammalian SIN3 complex and HDACs, and thereby affect the expression of specific genes. Results TIS7 is up-regulated and translocates into the nucleus of c-JunER cells losing epithelial polarity We were interested in the identification of genes whose expression changed as a result of activation of c-JunER. In the present study we performed DD RT–PCR with RNA samples isolated from untreated and 48 h β-estradiol (E2)-induced c-JunER cells. One of the several differentially expressed genes was tis7. A full-length TIS7 cDNA was subsequently cloned, which was identical to the one described in Varnum et al. (1989a). This nucleotide sequence encodes a protein of 449 amino acids that migrates on SDS–PAGE at ∼53 kDa. After 48 h of E2 treatment, the tis7 gene was up-regulated 5- to 10-fold (Figure 1A). Upon removal of E2, as the cells regain their epithelial polarity, the tis7 mRNA expression returned back to basal levels. Since estrogen hormones are potent mitogens for mammary epithelial cells, EpH4 parental cells of the c-JunER cell line were treated with an identical concentration of E2 as a control. The mRNA levels of the tis7 gene were not affected (Figure 1A, right panel). Figure 1.TIS7 mRNA and protein levels are up-regulated and TIS7 protein translocates into the nucleus of E2-activated c-JunER cells. (A) Northern blot analysis of total RNA isolated from c-JunER cells hybridized with a TIS7 cDNA probe. Control cells (−), 48 h E2-treated cells (+), reverted cells (Rev = cells grown for 48 h with E2 and then cultured for two additional passages in the absence of E2). Northern blot analysis of EpH4 parental cell line. One representative northern blot from five (c-JunER cells) or seven (EpH4 cells) independent experiments is shown. (B) Specificity of the affinity-purified TIS7 rabbit polyclonal antibodies. Left panel: GST–TIS7 fusion proteins detected by western blotting using α-GST antibodies. Middle panel: same proteins analysed using the affinity-purified α-TIS7 antibodies. Right panel: immunoprecipitation experiment using same the α-TIS7 antibodies without (−peptide) or after pre-incubation with the peptide used for immunization. HeLa cells were transfected with Xpr–TIS7 vector construct and equal amounts of cell lysate were used for immunoprecipitation. Immunoprecipitated protein Xpr–TIS7 was detected by western blotting using α-Xpress monoclonal antibodies. (C) Western blot analysis of TIS7 protein in control (−E2) and E2-treated (+E2) c-JunER cells, respectively. Equal loading is documented by the actin immunostaining (bottom). Western blot images were scanned, quantified and are presented as a ratio between the intensity of the specific TIS7 and actin input control signals (mean ± SD, n = 3). (D) Laser scanning confocal immunofluorescence analysis of TIS7 and ZO-1 localization in c-JunER polarized cells (−E2; left panel) and cells treated with 10−6 M E2 for 4 days (right panels). Loss of polarity of the epithelial cells was followed by measuring the transepithelial resistance (TER values). Size bar = 10 μm. Download figure Download PowerPoint We generated a rabbit polyclonal antiserum against a peptide comprising 18 N-terminal amino acid residues of TIS7. The TIS7 antiserum recognized the GST–TIS7 protein with the predicted size of 80 kDa on western blots (Figure 1B, middle panel). It did not cross-react with GST protein loaded in comparable amounts, as documented by the α-GST antibody blotting (Figure 1B, left panel). Next, we tested the affinity-purified α-TIS7 antibody in immunoprecipitations of lysates from HeLa cells transfected with the His6Xpr-TIS7 expression construct. The α-TIS7 antibody specifically immunoprecipitated the 63 kDa His6Xpr-TIS7 band recognized by the α-Xpress monoclonal antibody. The specificity of the immunoprecipitation was confirmed by its peptide inhibition (Figure 1B, right panel). In accordance with the DD RT–PCR results, TIS7 protein levels in total cell lysates were also up-regulated upon c-JunER activation (up to 3-fold) (Figure 1C, graph). TIS7 protein levels were induced 4 h following the onset of the E2 treatment and remained up-regulated for >48 h (Figure 1C). Effects of c-JunER activation on the subcellular distribution of TIS7 were analysed by confocal laser scanning immunofluorescence microscopy. In fully polarized cells [4 days in culture; epithelial polarity documented by the ring-like staining of the tight junction protein ZO-1 and high transepithelial electric resistance (TER) values], the majority of TIS7 localized in the vicinity of the lateral plasma membrane (Figure 1D, −E2). Upon c-JunER activation, cells lost epithelial polarity (as documented by the disrupted ZO-1 staining and low TER values), the lateral staining of TIS7 was reduced and the TIS7 protein accumulated in the nuclei (Figure 1D, +E2), similar to sparsely seeded undifferentiated cultures (data not shown). In summary, the loss of polarity caused by the activation of the c-JunER fusion protein in mouse mammary epithelial cells was accompanied by an increase in both tis7 mRNA and protein levels, and by translocation of the TIS7 protein into the nucleus. TIS7 overexpression changes the polarity of epithelial cells Next we examined whether overexpression of TIS7 protein itself would affect the polarity of the mammary epithelial cells. We constructed a recombinant TIS7 chicken adenovirus (CELO) and expressed TIS7 by infection of fully polarized monolayers of c-JunER cells. Virus expressing eGFP was used as a control. Immunofluorescence analysis revealed that the majority of TIS7-overexpressing cells lost the polarized epithelial phenotype, as assessed by the distribution of junctional proteins β-catenin and ZO-1, respectively (Figure 2A and B). These cells were higher, rounded up and protruded from the monolayer (Figure 2A and B, x–z section; note the difference in the thickness of the x–z section, reflecting the number of cell layers). The cumulative effect of prolonged TIS7 overexpression on the epithelial cell polarity also reflected the fall in the transepithelial resistance, as shown in the graph (Figure 2C). Although eGFP was overexpressed in similar amounts and also accumulated in the nuclei of infected cells, it did not affect epithelial cell polarity. We conclude that overexpression of TIS7 may have interfered with the polarized epithelial phenotype since contact with substratum and adhesion to neighboring cells were impaired. The nuclear localization of TIS7 in unpolarized cells suggested a role in the regulation of gene expression. Figure 2.TIS7-overexpressing cells lose epithelial polarity. (A) Cells were infected with CELO-recombinant viruses. Overexpressed TIS7 was detected by laser scanning confocal immunofluorescence microscopy using α-Myc antibody, endogenous β-catenin with the monoclonal antibody and eGFP directly. Top panels show sections through the x–y planes and underneath the x–z side view is shown. Bar = 10 μm. (B) TIS7-overexpressing cell grows on the top of the monolayer of otherwise polarized epithelial cells (arrowhead marks the filter insert). In polarized cells, tight junctional protein ZO-1 forms a typical ring-like structure (arrow). (C) TIS7 overexpression caused a significant decrease in the transepithelial resistance. Download figure Download PowerPoint Analysis of TIS7 effects on gene transcription using cDNA microarray analysis In order to find out whether nuclear TIS7 had the capacity to affect transcription, we performed a cDNA microarray analysis. The experimental set-up of this analysis is explained in detail in the Supplementary data (available at The EMBO Journal Online). The expression data were ranked using a web-based software tool developed in our laboratory to query a relational database (MicroarrayViewer; see Supplementary data). Ninety-three percent of genes were specifically down-regulated by TIS7 overexpression, while only two genes were up-regulated. The expression differences corresponded to an average expression ratio of 2.2. Transcriptional effects of TIS7 were independently, quantitatively, reconfirmed for several of the candidate genes using Real-Time PCR (Light Cycler™) (Table I). Western blot analysis also showed that protein levels of two candidate genes (CRABP II and OPN) were down-regulated in cells overexpressing TIS7 (Supplementary data). Table 1. Identification of genes that transcription specifically changed following the TIS7 overexpression by cDNA microarray analysis Sequence annotation UniGene accession No. Differential expression: TIS7–EGFP Real-Time PCR Cellular retinoic acid binding protein II (crabp II)* W81912 −4.1 −7.5 Osteopontin/secreted phosphoprotein I (spp1)* AA108928 −2.9 −2.1 w.s.t. CG5676 gene product # AA238637 −2.6 h.s.t. AF150733_1 AD-014 protein # W74865 −2.2 Unknown EST 34061 −2.1 h.s.t. NADH-ubiquinone oxireductase B14 SU # 62323 −2.1 m.s.t. 60S ribosomal protein L39 # AA178015 −2.1 h.s.t. 60S ribosomal protein L22 # AA174807 −2.0 Unknown EST W41563 −2.0 Myosin heavy polypeptide 8, skeletal muscle, perinatal* W13528 −1.9 Synapsin Ib* AA033398 −1.9 w.s.t. wdnm1 protein precursor # AA105830 −1.8 Feminization-1a homolog (fem-1a)* AA434902 −1.8 h.s.t. cystathione-γ-lyase # AA245993 −1.7 Emopamil binding protein (ebp)* W08348 −1.7 −2.8 Cathepsin Z* W14289 −1.7 Peptidylprolyl-isomerase A* AA499926 −1.7 Fibroblast inducible secreted protein 12 (fisp12)* W36541 −1.7 Pituitary tumor transforming gene protein 1 (pttg1)* A250500 −1.6 Thymidine kinase 1* AA041834 −1.6 Unknown EST W09957 −1.6 Homeobox, msh-like 2* AA016730 −1.5 DNA segment, Chr 15, Wayne State University 77 AA014127 −1.5 Torsin family 2, member A* AA190292 −1.5 Hexokinase 1* W11571 −1.5 h.s.t. SRB7 # AA067890 −1.5 Split hand/foot deleted gene 1* AA462396 −1.5 h.s.t. ubiquinol–cytochrome c reductase complex (7.2 kDa) # AA087137 −1.5 h.s.t. myelin protein zero (p0) precursor # AA097191 +1.5 +3.8 Intracisternal A-particle mRNA AA404014 +1.5 Using the MicroarrayViewer program (see Supplementary data), genes specifically regulated by ectopic TIS7 could be extracted from the vector associated and from the great majority of unaffected genes. Candidate genes are listed according to their level of differential expression. w.s.t., weakly similar to; m.s.t., moderately similar to; h.s.t., highly similar to; *, functionally annotated TIS7 target genes; #, TIS7 target genes of ‘uncertain’ identity. Confirmation of the cDNA microarray hybridization results by Real-Time PCR analysis. Values of TIS7-regulated gene expressions were normalized and then compared as a ratio of TIS7 sample values and eGFP sample values. In conclusion, the cDNA microarray experiment revealed that TIS7 overexpression preferentially caused down-regulation of a specific set of genes and this finding, together with TIS7 nuclear localization, suggest a potential role for TIS7 as a transcriptional co-repressor. TIS7 interacts with the SIN3 HDAC complex In order to identify the mechanism by which TIS7 affects transcription, we decided to identify proteins interacting with the mouse TIS7 protein. In a yeast two-hybrid screen, we used a mouse embryo cDNA library and the full-length cDNA (amino acids 1–449), as well as N-terminal (amino acids 1–227), C-terminal (amino acids 262–449) and central domains (amino acids 240–280) of TIS7 as baits. We identified mSin3B, a member of the SIN3 HDAC complex (four positive clones), as a protein potentially interacting with the N-terminal domain of the TIS7 protein. Having established the interaction in yeast two-hybrid screens, we tested whether the TIS7 interaction with mSin3B could also be demonstrated in vivo. First, HeLa cells were transfected with a FLAG-tagged mSin3B long form (FLAG-mSin3B LF) (Naruse et al., 1999) and immunoprecipitated using α-FLAG antibodies. Endogenous TIS7 protein co-immunoprecipitated with FLAG-mSin3B (Figure 3A). Figure 3.TIS7 interacts with mSin3B. (A) HeLa cells were transfected with FLAG-mSin3B LF expression plasmid. Cell lysates were immunoprecipitated with α-FLAG tag monoclonal antibody and co-immunoprecipitated endogenous TIS7 was detected by western blot analysis. (B) HeLa cells were co-transfected with N- or C-terminal Myc9-tagged TIS7 truncation mutants and FLAG-tagged mSin3B long-form construct as indicated. Cell lysates were immunoprecipitated with the antibodies against the Myc tag, and co-immunoprecipitated mSin3B was detected by immunoblotting using α-FLAG antibodies. The bottom panel shows TIS7-Myc9 truncation mutants immunoprecipitated with the α-Myc antibodies. (C) HeLa cells were co-transfected with C-terminally Myc9-tagged full-length TIS7 construct and different N-terminally FLAG-tagged mSin3B constructs (LF, long form; P, PAH domain; arrowheads point towards the Sin3 fusion protein bands, smaller bands are degradation products). Protein complexes were immunoprecipitated with α-FLAG tag antibodies. In the lower panel are mSin3B constructs immunoprecipitated with α-FLAG antibodies detected with the same antibody. Schemes under both panels depict expressed parts of the proteins and level of interaction (−, +, ++). Download figure Download PowerPoint Next, we mapped the mSin3B interaction domains on the TIS7 protein. FLAG-tagged mSin3B LF was transfected into HeLa cells either alone or together with a series of Myc9-tagged TIS7 deletion constructs and immunoprecipitations were performed using α-Myc antibodies. Two TIS7 constructs encompassing the N-terminal amino acids 1–321 and 1–367 co-immunoprecipitated mSin3B. However, a partially overlapping construct expressing the protein encoded by amino acid residues 140–449, lacking the N-terminal domain of the TIS7 protein, was not capable of binding mSin3B (Figure 3B). The results of this domain mapping experiment confirmed our initial finding that TIS7 interacted in the yeast two-hybrid screen with the mSin3B protein through its N-terminal domain. Thus, we concluded that the region necessary and sufficient for the interaction with mSin3B is the TIS7 sequence between the amino acids 1 and 140. Some of the specific domains of mSin3B important for its interaction with interacting partners are already well characterized, e.g. interaction of the paired amphipathic helix (PAH)2 domain with the transcription factor MAD (Ayer et al., 1996; Harper et al., 1996; Laherty et al., 1998), or of PAH1 and PAH3 domains with N-CoR (Alland et al., 1997; Heinzel et al., 1997). Therefore, we examined the in vivo interactions between the series of mSin3B deletion constructs and full-length TIS7. Among the series of mSin3B deletion constructs examined (Figure 3C), mSin3B LF as well as constructs containing the PAH2 domain and the intervening region between the PAH1 and PAH2 domains represented significant binding to TIS7. The PAH1 domain alone also bound TIS7, though less efficiently. In contrast, constructs containing the C-terminal half of mSin3B only, encompassing PAH3 through PAH4 domains (mSin3B P3-4), showed no interaction with TIS7 (Figure 3C). It was shown previously that mSin3 proteins (A and B), as part of the SIN3 complex, recruit HDACs to specific transcription factors (e.g. Mad or NRSF) and thereby repress transcription (Ayer et al., 1995, 1996; Korhonen et al., 1998; Naruse et al., 1999). To test whether TIS7 is associated with HDAC1 in vivo, TIS7 immunoprecipitates were assayed for HDAC1 by western blot analysis. TIS7 antibody specifically co-immunoprecipitated HDAC1 and formation of this precipitate was blocked with the cognate immunogen. Endogenous HDAC1 was co-immunoprecipitated in a dose-dependent manner with increasing amounts of the α-TIS7 antibody (Figure 4A). In the reciprocal experiment, endogenous TIS7 was co-immunoprecipitated with the monoclonal antibody against the endogenous HDAC1 (Figure 4B). To test whether TIS7 has the capacity to associate with other HDACs besides HDAC1, we performed in vitro pull-down assays using GST–TIS7 fusion protein. Radioactively labelled HDAC1, HDAC2 and HDAC3 specifically interacted with the GST–TIS7 protein (Figure 4C). Similarly, GST–TIS7 interacted with N-CoR nuclear receptor co-repressor, a component of HDAC complexes. These interactions were specific since, first, products of in vitro coupled transcription/translation reaction did not interact with GST control protein alone and, secondly, GST–TIS7 did not interact with an unrelated nuclear protein, human cohesin subunit SCC1, synthesized in an identical way (Figure 4C). Figure 4.TIS7 interacts with HDAC1. (A) HeLa cell lysates were immunoprecipitated with the affinity-purified rabbit α-TIS7 antibodies, without (−) or with (+) pre-incubation with the peptide used to raise the TIS7 antibodies (left panel). Co-immunoprecipitated HDAC1 was detected by western blotting. Increasing amounts of the α-TIS7 antibodies co-immunoprecipitated increasing amounts of the endogenous HDAC1. Bottom panels show immunoprecipitated endogenous TIS7 detected by western blotting using the same α-TIS7 antibodies. (B) In a vice versa experiment, HeLa cell lysates were immunoprecipitated with the monoclonal antibodies against HDAC1 and co-immunoprecipitated endogenous TIS7 was detected by western blotting (−Ab, pre-immune serum). The bottom panel documents amounts of immunoprecipitated endogenous HDAC1. (C) GST pull-down analysis using recombinant GST–TIS7 protein. 35S-labelled HDAC1, HDAC2, HDAC3, hSCC1 and N-CoR products of in vitro coupled transcription/translation reaction in rabbit reticulocyte lysate were incubated with GST proteins bound to the glutathione beads, washed and eluted with sample buffer. Bound proteins were analysed by autoradiography. Equal loading of GST and GST–TIS7 proteins was confirmed by Coomassie Blue staining (bottom panel). Download figure Download PowerPoint In the next experiment, we tested whether TIS7 may associate with some of the currently known subunits of the SIN3 complex in mouse epithelial c-JunER cells. We analysed c-JunER nuclear extract fractions after separation by continuous sucrose gradients. In E2-untreated cells, part of the TIS7 pool co-sedimented with mSin3B, HDAC1 and SAP30 during sucrose gradient centrifugation (Figure 5A, fraction 9). Another part of the TIS7 pool in these normally polarized cells appeared in lighter fractions from the gradient centrifugation representing monomers. However, following 4 days treatment of c-JunER cells with E2, there was a significant increase in the amount of TIS7 that co-sedimented with mSin3B and HDAC1 during sucrose gradient centrifugation (fraction 10). At the same time, TIS7 sedimenting in the lightest fractions of the sucrose gradient (fractions 2 and 3) disappeared. We have confirmed that TIS7 associates specifically with the SIN3 complex in an independent sucrose gradient separation of a nuclear extract by blotting with antibodies against MTA2, a component of a different HDAC complex. MTA2 fractionated in different fractions than TIS7 (data not shown). Figure 5.TIS7 can associate with the SIN3 complex. (A) mSin3B, HDAC1, TIS7 and SAP30 partially co-fractionate. Nuclear extracts prepared from control (−E2) or 4 day E2-treated c-JunER cells (+E2) were fractionated on a 10–